Change in structure of the N-terminal region of transthyretin produces change in affinity of transthyretin to T4 and T3

The relationship between the structure of the N-terminal sequence of transthyretin (TTR) and the binding of thyroid hormone was studied. A recombinant human TTR and two derivatives of Crocodylus porosus TTRs, one with the N-terminal sequence replaced by that of human TTR (human/crocTTR), the other with the N-terminal segment removed (truncated crocTTR), were synthesized in Pichia pastoris. Subunit mass, native molecular weight, tetramer formation, cross-reactivity to TTR antibodies and binding to retinol-binding protein of these recombinant TTRs were similar to TTRs found in nature. Analysis of the binding affinity to thyroid hormones of recombinant human TTR showed a dissociation constant (Kd) for triiodothyronine (T3) of 53.26 ± 3.97 nm and for thyroxine (T4) of 19.73 ± 0.13 nm. These values are similar to those found for TTR purified from human serum, and gave a K d T3/T4 ratio of 2.70. The affinity for T4 of human/crocTTR (K d = 22.75 ± 1.89 nm) was higher than those of both human TTR and C. porosus TTR, but the affinity for T3 (Kd = 5.40 ± 0.25 nm) was similar to C. porosus TTR, giving a Kd T3/T4 ratio of 0.24. A similar affinity for both T3 (Kd = 57.78 ± 5.65 nm) and T4 (Kd = 59.72 ± 3.38 nm), with a Kd T3/T4 ratio of 0.97, was observed for truncated crocTTR. The obtained results strongly confirm the hypothesis that the unstructured N-terminal region of TTR critically influences the specificity and affinity of thyroid hormone binding to TTR.